Eukaryotic protein kinases catalyze the phosphorylation of tyrosine, serine, and threonine residues and regulate essentially all cellular processes. Protein kinases are therefore important therapeutic targets for a variety of human diseases. The human genome encodes approximately 500 protein kinases, all of which share a highly conserved ATP binding site. Nearly all known small-molecule kinase inhibitors target this site, a deep hydrophobic cleft containing all of the essential catalytic residues. Thus, a central problem in the chemical biology of protein kinases concerns the development of selective inhibitors that discriminate among these similar binding sites. This proposal describes a structural bioinformatics approach for the design of selective, irreversible kinase inhibitors. Beginning with a sequence alignment of all protein kinase domains in the human genome, we identified a small subset that possess a cysteine residue in a unique location within the ATP binding site. The location of this cysteine, predicted by our analysis of multiple crystal structures, forms the starting point for the design of novel, electrophilic inhibitors. We hypothesize that an electrophilic substituent appended to an appropriate scaffold will rapidly alkylate the cysteine, thereby blocking the ATP binding site and irreversibly inhibiting the enzyme. The long-term goal of this project is to develop highly selective, cell-permeable inhibitors to unravel the precise cellular roles of these protein kinases, thought to regulate processes as diverse as transcription, apoptosis, chromosome segregation, and cytokinesis.